A phenomenon of energy transfer which occurs between a light-emitting enzyme and a fluorescent protein is a natural phenomenon which occurs in luminescent jellyfishes and luminescent mushrooms, and a mechanism thereof has been elucidated at a molecular level (Ohmiya, Y. and Hirano, T: Shining the light: the mechanism of the bioluminescence reaction of calcium-binding photoproteins. Chemistry & Biology 3:337-347, 1996). Furthermore, a Renilla luciferase and green fluorescent protein fusion gene which simulates this natural phenomenon has been constructed, and a method for quantitatively and qualitatively monitoring the gene expression using a luciferase activity and a fluorescent activity has been known (U.S. Pat. No. 5,976,796 and WO98/14605).
The Renilla luciferase is an enzyme purified from Renilla reniformis. This enzyme catalyzes oxidative decarboxylation of a luminescent substrate, coelenterazine in the presence of the enzyme to produce blue light with a maximum wavelength of 478 nm. However, in the natural world, the light shifts to green light having the maximum wavelength of 510 nm attributed to the energy transfer to a green fluorescent protein present in Renilla reniformis. A Renilla luciferase gene has been already cloned, and a cDNA thereof has been shown to be useful as a reporter gene for measuring a transcription activity of a gene.
The fluorescent protein sometimes coexists with the light-emitting enzyme as those in the luminescent jellyfish and luminescent Renilla whereas it sometimes singly exists as that in cactuses. A green fluorescent protein purified from the luminescent jellyfish, Aequorea victoria receives blue light from a photoprotein, and converts this to green light. This gene has been cloned, and is a strong reporter gene in various biological systems (bacterial, fungal and mammalian tissues) because green fluorescence is emitted by blue excitation light without need of a cofactor when a cDNA thereof is expressed in a cell. In modified types of a wild-type green fluorescent protein, there are a modified one having a shift toward a red with blight luminescence and a modified one whose stability is improved in mammalian cells. A cDNA of a red fluorescent protein has been also cloned from a naturally occurring coral, and is also useful as the reporter gene.
A construct of the Renilla luciferase and green fluorescent protein fusion gene previously invented is distributed in an entire cytoplasm depending on a character of Renilla luciferase, and is not localized. A luminescent substrate of the Renilla luciferase has no cell permeability, and the gene expression can not be detected unless the cells are once lysed.
A luminescent crustacean, marine ostracod, Vargula hilgendorfii and its related species Cypridina noctiluca have a secretory light-emitting enzyme, and a cDNA of the Vargula luciferase has been already cloned. It reacts with a marine ostracod luminescent substrate, Cypridina luciferin to emit blue light with a maximum luminescent wavelength of 460 nm. A gene transcription activity can be measured without lysing the cells because the cloned cDNA works as the reporter gene and the light-emitting enzyme is secreted out of the cells. The secretion of a protein from the cells can be visualized by image-analyzing of this secretory light-emitting enzyme. Meanwhile, it emits the blue light, but it has been never practically applied as a donor protein of energy transfer.
It is an object of the present invention to construct a secretory or membrane-binding protein fusion having an energy transfer property, make and use a construct having two functions of an energy-generating protein and an energy-receiving protein measurable out of a cell or on a cell surface. A gene transcription activity can be measured out of the cell as the energy transfer property, and together, a secretory pathway from an inside to an outside of the cell can be evaluated by the energy transfer property. A peptide three dimensional structure information can be obtained by inserting a monitor peptide between the energy-generating protein and the energy-receiving protein or inside the energy-generating protein or the energy-receiving protein of the secretory or membrane-binding chimeric protein, and using a change of the energy transfer by cutting the monitor peptide as an indicator. In particular, when a light-emitting enzyme and a fluorescent protein are used as the energy-generating protein and the energy-receiving protein, a fusion construct capable of measuring fluorescence without using excitation light can be made because the fluorescent protein is excited with the light emitted from the light-emitting enzyme and a color of the emitted light is changed by the energy transfer.